The invention relates to various yeast strains of the type Yarrowia lipolytica as well as relevant methods for the biocatalytic preparation of ω-hydroxy fatty acids or dicarboxylic acids with the aid of these strains.
Dicarboxylic acids comprise carboxylic acids having two carboxy groups (general structure: HOOC—(CH2)n—COOH), which have diverse applications in the chemical industry, e.g. for the preparation of fragrances, adhesives, Nylon and other polyamides, resins, corrosion inhibitors or lubricants. In particular, the biotechnological production of long-chain dicarboxylic acids is of particular interest since a number of undesired side products are formed during the chemical synthesis of the same.
ω-hydroxy fatty acids contain at the Cω atom (C atom having the furthest possible distance from the carboxyl group) a hydroxyl group (general structure: HOOC—(CH2)n—CH2OH). ω-hydroxy fatty acids are also of great importance in the chemical industry since they are used, inter alia, in lubricants, adhesives, cosmetics and cancer therapeutic agents. Furthermore, ω-hydroxy fatty acids could acquire great importance in future since they can be used as monomers for the synthesis of bioplastics.
In order to obtain ω-hydroxy fatty acids and dicarboxylic acids from n-alkanes and fatty acids, the non-conventional yeast Yarrowia (Y.) lipolytica is to be used within the framework of the invention. The yeast Y. lipolytica was selected as host organism for the bioconversion since it is able to use a plurality of substrates as carbon source. In addition to glucose, glycerol, proteins, alcohols and acetate, this also includes a plurality of hydrophobic substrates such as vegetable oils, fats, fatty acids and n-alkanes (Barth G & Gaillardin C (1997) FEMS Microbiol Rev 19: 219-237). The hydrophobic substrates are emulsified by the yeasts and assimilated into the cell interior with the aid of specialized membrane transporters (Fickers P, Benetti P H, Waché Y, Marty A, Mauersberger S, Smit M S & Nicaud J M (2005) FEMS Yeast Res 5: 527-543). n-alkanes assimilated into the cell are converted stepwise to fatty acid having the same chain length (FIG. 1) in the course of the primary (monoterminal) alkane oxidation. The fatty acids are then broken down in the course of the β-oxidation to acetyl CoA which flows into the tricarboxylic acid and the glyoxylate cycle. In parallel with the β-oxidation of fatty acids in the peroxisomes, ω-oxidation takes place in the endoplasmic reticulum (cf. FIG. 1). However, this diterminal oxidation of fatty acids naturally takes place to a far lesser extent than the β-oxidation.
Various yeasts such as, for example, Candida (C.) tropicalis and Y. lipolytica are capable of converting long-chain alkanes or fatty acids to α,ω-dicarboxylic acids. In order that the hydrophobic substrates can be converted at all to the respective dicarboxylic acid, their metabolization in the course of the β-oxidation must be prevented. This can be accomplished, for example, by deletion of the POX gene coding for the acyl-CoA-oxidase. If the β-oxidation is eliminated, this leads to an increased ω-oxidation of the fatty acids (Smit et al. (2005) Biotechnol. Lett. 27: 859-864).
The microbial production of long-chain dicarboxylic acids has already been carried out with the aid of the yeast C. tropicalis:                 Picataggio S, Rohrer T, Deanda K, Lanning D, Reynolds R, Mielenz J & Eirich L D (1992) Biotechnology (NY) 10: 894-898.        
In this case, the production of the dicarboxylic acids was primarily achieved by deletion of the genes POX4 and POX5. There are a number of patents which protect corresponding C. tropicalis production strains and relevant production methods (e.g. U.S. Pat. No. 4,339,536A, EP296506A2 and WO200017380A1).
In a 2013 patent specification it is described how yeasts in general (in a manner known per se) can be induced to produce increased quantities of dicarboxylic acids. In the exemplary embodiments however, only C. tropicalis is specifically discussed here (WO201306730A2, WO201306733A2).
The microbial production of long-chain ω-hydroxy fatty acids has also already been carried out with the aid of the yeast C. tropicalis:                 Lu W, Ness J E, Xie W, Zhang X, Minshull J & Gross R A (2010) J Am Chem Soc 132: 15451-15455.        
For this purpose, in addition to the genes POX4 and POX5, six cytochrome P450, four alcohol oxidase and six alcohol dehydrogenase genes were deleted. These strains and production methods are already protected under patent law (WO2011008232A2).
Furthermore, it has already been described how Y. lipolytica can be induced to form dicarboxylic acids:                Smit M S, Mokgoro M M, Setati E & Nicaud J M (2005) Biotechnol Lett 27: 859-864.        
In this case, all the presently known acyl-CoA-oxidase genes (POX1, POX2, POX3, POX4, POX5 and POX6) as well as the acyl-CoA-diacylglycerol-acyltransferase gene (DGA1) and the lecithin-cholesterol-acyltransferase gene (LRO1) were deleted. Furthermore, the NADPH-cytochrome P450-reductase gene (CPR1) was overexpressed (WO200664131A8).
In addition to the biotechnological production of ω-hydroxy fatty acids and dicarboxylic acids with the aid of genetically modified yeasts using the intracellular ω-oxidation of fatty acids, there are also other biotechnological methods based on other enzymatic conversions. An example of this is the use of Baeyer-Villiger monooxygenase, which catalyzes the conversion of a ketone to the ester (WO2013151393A1).